We are investigating the structural biology of chromatin. Our overreaching goal is to refine the overall view of chromatin's architecture by understanding how the nucleosome interfaces with the cellular machinery based on sequence variations in its own proteins or interactions with outside molecules.

Our long-term goal research is to investigate the structural properties of chromatin, and to understand how transcription, replication, recombination, and repair take place within the context of highly compacted chromatin. We are particularly interested in mechanistic and structural aspects of these fundamental questions. We are using multipronged approaches including x-ray crystallography, small-angle x-ray scattering, fluorescence resonance energy transfer, analytical ultracentrifugation, and atomic force microscopy, conventional biochemistry, molecular biology, and yeast genetics to investigate fundamental questions of chromatin structure control.

Interaction of Nucleosomes with Transcription Factors and other cellular proteins. The nucleosome is the elemental repeating unit in chromatin, consisting of two copies each of the four histone proteins (the histone octamer) around which 146 base pairs of DNA are wrapped in nearly two turns of a tight superhelix. Using a combination of methods, most notably x-ray crystallography and fluorescence resonance energy transfer, we investigate the structural determinants and the structural changes that are inflicted upon the nucleosome and the interacting protein upon binding to nucleosomal DNA. Effects on higher-order structure are also studied using analytical ultracentrifugation and atomic force microscopy. Several of the factors under investigation are of clinical importance, either as targets for anti-cancer drugs, or because mutations in the corresponding gene are correlated with certain diseased states.

For example, by studying how Kaposi's sarcoma herpesvirus protein LANA (latency-associated nuclear antigen) enables the viral genome to tether onto chromosomes so that virus is not lost from cells, we found that LANA engages histones H2A and H2B to dock onto chromosomes by binding to the nucleosomal surface via a tight hairpin motif. This study (which is the result of an ongoing collaboration with Kenneth Kaye [Harvard Medical School]) unequivocally demonstrates how a highly structured nucleosomal surface acts as an interaction platform for molecular recognition.

Nucleosome Assembly and Histone Exchange Eukaryotic chromatin is highly dynamic and turns over rapidly in the absence of DNA replication and transcription. Acidic histone chaperones such as Nucleosome Assembly Protein 1 (NAP1) are implicated in this process. Although initially identified as histone chaperones and chromatin assembly factors, additional functions include roles in tissue-specific transcription regulation, apoptosis, histone shuttling, cell cycle regulation etc., and thus extend beyond those of a simple chaperone and assembly factor. Some family members are essential in mammals due to an as yet uncharacterized role in neuronal development. Several have been characterized as oncoproteins, and many have been found in complex with enzymes that post-translationally modify histones, or are implicated in ATP-dependent chromatin remodeling.

In vitro, NAP1 reversibly removes and replaces H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange and nucleosome sliding. The significance of these functions is being investigated in vivo. The crystal structure of yeast NAP1 is the first for a NAP family member and reveals a novel fold with implications for histone transport and exchange. The structure suggested several hypotheses regarding the function of the NAP1 protein family that are now being tested by a variety of methods, for example, small angle x-ray scattering, FRET, and in vivo studies (These studies are also supported by a grant from the National Institutes of Health.)

Histone Variants The replacement of canonical histones with histone variants has emerged as an important pathway to alter the biochemical makeup of chromatin locally, with the potential to exert considerable influence on the structure and function of chromatin. Histone variants are distinct nonallelic forms of conventional, major-type histones that form the bulk of nucleosomes during replication and whose synthesis is tightly coupled to S phase. They are found in most eukaryotic organisms and are expressed in all tissue types. We investigate the structures of variant nucleosomes and the effect of histone variant incorporation on higher order structure formation, and also test the hypothesis that specific histone chaperones are involved in their dynamic exchange.